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A novel method that enables antibody fragments to be immobilized on a sensor substrate with a high binding capability using molecular recognition has been developed. Using genetic engineering, we fabricated bispecific recombinant antibody fragments, which consist of two kinds of antibody fragments: a gold antibody fragment and a target molecule antibody fragment. Surface plasmon resonance (SPR) analysis indicated that these gold-binding bispecific antibody fragments bind directly to the gold substrate with high affinity ([K.sub.D] ~ [10.sup.-9] M). About 70% of the bispecific antibody fragments immobilized on the gold substrate retained their target protein-binding efficiency. The Sips isotherm was used to assess the heterogeneity in antibody affinity for the bispecific antibody fragments. The results showed that the immobilized bispecific antibody fragments exhibited an increased homogeneity of affinity ([K.sub.D]) to target molecules when compared with monospecific antibody fragments immobilized by conventional methods. The use of bispecific antibody fragments to directly immobilize antibody fragments on a solid-phase substrate offers a useful platform for immunosensor applications. 10.1021/ac100557k

A membrane protein on the surface of a single living mammalian cell was imaged by scanning electrochemical microscopy (SECM). The epidermal growth factor receptor (EGFR) is one of the key membrane proteins associated with cancer. It elicits a wide range of cell-type-specific responses, leading to cell proliferation, differentiation, apoptosis, and migration. To estimate EGFR expression levels by SECM, EGFR was labeled with alkaline phosphatase (ALP) via an antibody. The oxidation current of PAP (p-aminophenol) produced by the ALP-catalyzed reaction was monitored to estimate the density of cell surface EGFR. EGFR measurement by SECM has three advantages. First, a single adhesion cell can be measured without peeling it from the culture dish; second, it is possible to optimize labeling antibody concentrations by using living cells because detection of faradaic current is suitable for quantitative estimation in situ; and third, SECM measurements afford information on the expression state at the cell membrane at the single-cell level. In this study, we optimized the concentration of labeling antibody for EGFR at the cell surface and confirmed distinct differences in EGFR expression levels among three types of cells. SECM measurements were compatible with the results of flow cytometry.

A comparaison of the inhibitory activity of NH2-terminal human cysteine proteinase fused with porcine adenylate kinase shows that the amino-terminal is necessary for its activity. Removal of six residues from the amino-terminal decreases the inhibitory activity while removal of 15 residues completely inactivates the inhibitor. However the removal of the Met residue from the amino-terminal or substitution of the Pro3 with Leu residue does not affect the inhibitory activity. Substitution of the Gky75 with His residue decreases the loss of activity.

Quantum chemical methods are used to determine geometry, reorganization energy, and vibrational and electronic spectra of pigment molecules in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides. It was found that some low-energy vibrations, particularly those of ring side group affects the charge-transfer excited state and hence very likely are involved in the charge-separation process.

Two-dimensional exchange spectroscopy (EXSY) experiments are used to evaluate the solvent dependence of the stability of the bacteriochlorophyll (BChl) c dimer. It was found that antiparallel BChl c dimers show two exciton components for both Q(sub x)(0-0) and Q(sub y)(0-0) transition.

Recent advances in biotechnology have enabled the generation of antibodies with high affinity for the surfaces of specific inorganic materials. Herein, we report the synthesis of functional materials from multiple nanomaterials by using a small bispecific antibody recombinantly constructed from gold-binding and ZnO-binding antibody fragments. The bispecific antibody-mediated spontaneous linkage of gold and ZnO nanoparticles forms a binary gold-ZnO nanoparticle composite membrane. The relatively low melting point of the gold nanoparticles and the solubility of ZnO in dilute acidic solution then allowed for the bottom-up synthesis of a nanoporous gold membrane by means of a low-energy, low-environmental-load protocol. The nanoporous gold membrane showed high catalytic activity for the reduction of p-nitrophenol to p-aminophenol by sodium borohydride. Here, we show the potential utility of nanoparticle pairing mediated by bispecific antibodies for the bottom-up construction of nanostructured materials from multiple nanomaterials.

We report the observation of kinesin driven quantum dots (QDs) trapped in a microtubule loop, allowing the investigation of moving QDs for a long time and an unprecedented long distance. The QD conjugates did not depart from our observational field of view, enabling the tracking of specific conjugates for more than 5 min. The unusually long run length and the periodicity caused by the loop track allow comparing and studying the trajectory of the kinesin driven QDs for more than 2 full laps, i.e., about 70 μm, enabling a statistical analysis of interactions of the same kinesin driven object with the same obstacle. The trajectories were extracted and analyzed from kymographs with a newly developed algorithm. Despite dispersion, several repetitive trajectory patterns can be identified. A method evaluating the similarity is introduced allowing a quantitative comparison between the trajectories. The velocity variations appear strongly correlated to the presence of obstacles. We discuss the reasons making this long continuous travel distances on the loop track possible.

As a complementary tool to nanofluidics, biomolecular-based transport is envisioned for nanotechnological devices. We report a new method for guiding microtubule shuttles on multi-walled carbon nanotube tracks, aligned by dielectrophoresis on a functionalized surface. In the absence of electric field and in fluid flow, alignment is maintained. The directed translocation of kinesin propelled microtubules has been investigated using fluorescence microscopy. To our knowledge, this is the first demonstration of microtubules gliding along carbon nanotubes.

The grating substrate covered with a metal layer, a plasmonic chip, and a bispecific antibody can play a key role in the sensitive detection of a marker protein with an immunosensor, because of the provision of an enhanced fluorescence signal and the preparation of a sensor surface densely modified with capture antibody, respectively. In this study, one of the tumor markers, a soluble epidermal growth factor receptor (sEGFR), was selected as the target to be detected. The ZnO- and silver-coated plasmonic chip with precise regularity and the appropriate duty ratio in the periodic structure further enhanced the fluorescence intensity. As for sensor surface modification with capture antibody, a bispecific antibody (anti-sEGFR and anti-ZnO antibody), the concentrated bispecific antibody solution was found to nonlinearly form a surface densely immobilized with antibody, because the binding process of a bispecific antibody to the ZnO surface can be a competitive process with adsorption of phosphate. As a result, the interface on the plasmonic chip provided a 300× enhanced fluorescence signal compared with that on a ZnO-coated glass slide, and therefore sEGFR was found to be quantitatively detected in a wide concentration range from 10 nM to 700 fM on our plasmonic surface.

Antibodies, with their high affinity and specificity, are widely utilized in the field of protein engineering, medicinal chemistry, and nanotechnology applications, and our recent studies have demonstrated the recognition and binding of antibody for the surface on inorganic material. In this study, we generated a high-affinity gold-binding antibody fragment by a combination of peptide-grafting and phage-display techniques and showed the availability of the material-binding fragment for one-pot functionalization of nanoparticles as interface molecules. After a gold-binding peptide sequence was grafted into one of the complementarity determining regions of a single variable domain of a heavy-chain camel antibody, a combinatorial library approach raised by 20 times the affinity of the peptide-grafted fragment. The high-affinity gold-binding fragment (E32) spontaneously adsorbed on gold nanoparticles, and consequently the nanoparticles formed a stable dispersion in a high-ionic-strength solution. Multivalent and bispecific antibodies constructed on the E32 platform by means of fusion technology functionalized gold nanoparticles in one pot, and these functionalized nanoparticles could be used to obtain surface plasmon resonance scattering images of cancer cells and to spontaneously link two different nanomaterials. Here, we propose the bispecific antibodies as convenient interface molecules in the nanosized world.

Modular bispecific antibodies (BsAb's) that interact directly with a gold surface were engineered for immobilization on biosensing devices. The BsAb's consist of the variable fragments of antigold and antilysozyme antibodies connected via one of three linkers derived from naturally occurring proteins. The BsAb's were bound tightly to both the gold surface and to lysozyme, thus functioning as interface molecules between lysozyme and the gold surface without a substantial loss of antigen-binding activity. The antigen-binding capacity (the ratio of the amount of immobilized lysozyme to the amount of immobilized BsAb) on the gold surface reached 82%. An analysis of the correlation between binding capacity and linker characteristics indicated that the presence of a long, rigid linker sequence derived from a cellulase resulted in a higher antigen-binding capacity than did the presence of a long but relatively flexible glycine-rich linker. This result suggests a strategy for designing linkers suitable for BsAb-based biomolecular immobilization.

Antibodies with high affinity for the surface of a solid material would be advantageous in biomaterial science as a protein device. A human antibody fragment that binds to poly(hydroxybutyrate) (PHB), a biodegradable polymer matter, was generated by a phage display system. Clone PH7-3d3 was isolated after several rounds of selection and prepared as a fragment of immunoglobulin variable regions (Fv). The quartz crystal microbalance technique showed that PH7-3d3 Fv completely inhibited PHB enzymatic degradation by competing with PHB depolymerase. Kinetic analysis based on surface plasmon resonance demonstrated that PH7-3d3 Fv bound to the PHB film with an equilibrium dissociation constant of 14 nM. The three-dimensional structure of PH7-3d3 Fv was resolved to 1.7 A, revealing that the complementarity determining regions (CDRs) in the Fv fragment form a relatively flat surface on which uncharged polar and aromatic amino acids are distributed in clusters. The structure of PH7-3d3 Fv was similar to that of PHB depolymerase in the orientation of aromatic residues in the binding sites. Alanine scanning mutagenesis demonstrated that these aromatic residues, especially tryptophan residues in CDRs, were critical in the interaction between PH7-3d3 Fv and PHB. Our results suggest the possible selection of an antibody fragment that binds a material surface in a manner similar to protein-ligand interaction.